CXCR4与IL-35联合基因修饰间充质干细胞对溃疡性结肠炎局部免疫平衡的调节及清热燥湿凉血方的协同作用

Infusion of MSCs to treat ulcerative colitis has shown attractive prospect. However, insufficient homing of MSCs reduces its efficacy. So promoting homing of MSCs and enlarge its immunomodulatory effects on inflammatory topical focus are two core issues to be solved in the MSCs treatment of ulcerative colitis. Previous studies have suggested that the SDF-1/CXCR4 axis plays an important role in the regulation of homing of MSCs. IL-35 is an important cytokine that can regulate the differentiation of Treg and has function of immune suppression. Preliminary studies have been successfully constructed CXCR4, IL-35-transfected MSCs and could stably express CXCR4 and IL-35. Upregulating the expression of CXCR4 could promote MSCs homing to inflammatory colon. Based on this we designed: To make MSCs overexpress CXCR4 and IL-35 by joint genetically modifying. And use Qingrezaoshiliangxue formula to synergistically promote MSCs homing to inflamed intestinal tract. Thus MSCs could high express IL-35 in inflammatory topical focus and exert immunomodulatory and repair function. And the therapeutic effect of MSCs will be amplified. This study will provide a theoretical reference for effective use of MSCs and Qingrezaoshiliangxue formula to treat UC.

输注间充质干细胞(MSCs)治疗溃疡性结肠炎(UC)显示出诱人前景，然而存在MSCs归巢不足等瓶颈问题，因此如何促进MSCs向UC肠道归巢，并放大其对炎症局部免疫调节作用，是MSCs治疗UC亟待解决的两个核心问题。现已知SDF-1/CXCR4信号轴决定MSCs归巢能力,IL-35是近年发现调控Treg分化、具有免疫抑制功能的重要细胞因子。前期研究已构建CXCR4、IL-35转染的MSCs并能高表达CXCR4、IL-35，并发现上调CXCR4可促进MSCs向UC肠道归巢。基于此我们设计：对MSCs进行联合基因修饰使其过表达CXCR4和IL-35，上调CXCR4并采用清热燥湿凉血方协同增加MSCs向UC肠道靶向归巢，归巢的MSCs在炎症局部高表达IL-35，发挥免疫调节及修复功能，从而放大MSCs的治疗作用。本研究将为临床有效利用 MSCs及清热燥湿凉血方治疗UC提供理论依据。

输注间充质干细胞(MSCs)治疗溃疡性结肠炎(UC)显示出诱人前景，然而存在MSCs归巢不足等瓶颈问题，因此如何促进MSCs向UC肠道归巢，并放大其对炎症局部免疫调节作用，是MSCs治疗UC亟待解决的两个核心问题。现已知SDF-1/CXCR4信号轴决定MSCs归巢能力,IL-35是近年发现调控Treg分化、具有免疫抑制功能的重要细胞因子。前期研究已构建CXCR4、IL-35转染的MSCs并能高表达CXCR4、IL-35，并发现上调CXCR4可促进MSCs向UC肠道归巢。基于此我们设计：对MSCs进行联合基因修饰使其过表达CXCR4和IL-35，上调CXCR4并采用清热燥湿凉血方协同增加MSCs向UC肠道靶向归巢，归巢的MSCs在炎症局部高表达IL-35，发挥免疫调节及修复功能，从而放大MSCs的治疗作用。本课题利用体内、外实验阐述干细胞在其中的作用及相关机制。（一）体外分离、培养和鉴定骨髓间充质干细胞，分别用CXC4和IL-35慢病毒感染骨髓间充质干细胞。（二）利用Transwell技术检测慢病毒感染骨髓间充质干细胞的迁移能力，相同条件下CXCR4+IL-35-BMSCs迁移的细胞明显比空载的骨髓间充质干细胞迁移的细胞量更多（68.4vs18.2）（三）磁珠分选大鼠脾脏原代CD4+T细胞，流式细胞术检测基因修饰的骨髓间充质干细胞与CD4+T细胞共培养、骨髓间充质干细胞与CD4+T细胞共培养、CD4+T细胞， CD4+T细胞向Th17细胞及调节性T细胞的分化的影响。结果CXCR4+IL-35-BMSCs处理能够显著抑制CD4+T细胞中Th17细（0.30% vs. 1.72%），促进CD4+T细胞中Treg细胞（17.4% vs. 10.2%）。四、CXCR4-IL-35-BMSCs对大鼠溃疡性结肠炎的治疗作用及清热燥湿凉血方的协同作用；1、大鼠溃结模型的建立，2、动物分组处理，3、疗效评估及肠系膜和脾脏的Treg分化表达情况。综上结果提示在体外细胞实验中，基因修饰的骨髓间充质干细胞能够通过影影响骨髓间充质干细胞的迁移及CD4+T细胞向Th17及Treg细胞分化及相应细胞因子水平的改变而发挥调节免疫的作用.